Down's syndrome, also referred to as trisomy 21, is characterized by an extra copy of chromosome 21. People afflicted with Down's syndrome have severe mental retardation, reduced life expectancies, and abnormal immune responses that predispose them to serious infections as well as thyroid autoimmunity. Further, 40% of Down's syndrome patients have congenital heart disease and a 10 to 20-fold increased risk of developing leukemia relative to the general population. All Down's syndrome patients older than 40 develop neuropathological changes that are characteristic of Alzheimer's disease.
Prenatal tests to detect aneuploidy, such as trisomy 21, by amniocentesis or chorionic villus sampling (CVS) have been available since the late 1960s. Amniocentesis is the most common invasive prenatal diagnostic procedure. In amniocentesis, amniotic fluid is sampled by inserting a hollow needle through the mother's anterior abdominal and uterine walls into the amniotic cavity by piercing the chorion and amnion. It is usually performed in the second trimester of pregnancy. CVS is performed primarily during the first trimester, and involves collecting cells from the chorion which develops into the placenta.
Another invasive prenatal diagnostic technique is cordocentesis or percutaneous umbilical cord blood sampling, commonly known as fetal blood sampling. Fetal blood sampling involves obtaining fetal blood cells from vessels of the umbilical cord, and is performed about the 20th gestational week.
Amniocentesis is used selectively because it presents a risk of about 1% of inducing spontaneous abortion. CVS and fetal blood sampling carry a similar or higher risk of inducing abortion, and there is also concern that these procedures may lead to fetal limb malformations in some cases. Thus, amniocentesis, CVS and fetal blood sampling are procedures that are only employed if a pregnancy is considered at high risk for a serious congenital anomaly. Thus, some means is required to select those pregnancies that are at a significant risk of Down's syndrome to justify the risks associated with invasive prenatal diagnostic procedures, such as amniocentesis, CVS and fetal blood sampling.
Human chorionic gonadotropin (hCG) is a glycoprotein with 8 oligosaccharide side chains. Sugar residues account for approximately 30% of the molecular weight of hCG, and variation in oligosaccharide branching is a key factor in the hCG structure (Elliott et al., 1997, Endocrine 7:15-32). hCG exists in maternal urine in various forms including hyperglycosylated hCG, also called invasive trophoblastic antigen (ITA). While hCG is produced by differentiated syncytiotrophoblast cells, ITA is produced solely by invasive cytotrophoblast cells (Kovalevskaya et al., 2002, Mol Cell Endocrinol 194:147-55; Lei et al., 1999, Troph Res 13:147-59). ITA, which is the predominant form of hCG produced in invasive trophoblast disease and early pregnancy at the time of and following implantation, contains additional antennae on the oligosaccharide side chains (O'Connor et al., 1998, Prenat Diagn 18:1232-40). Each of the side chain antennae normally ends with a sialic acid residue. The extent of sialic acid content on the ITA molecule is dependent on abundance of sialic acid in the body. Phosphoenolpyruvate, which is a major intermediate in sugar metabolism, is a substrate in the production of sialic acid (Elliott et al., 1997, Endocrine 7:15-32). Thus, depending on cellular growth and metabolism, ITA can vary greatly in sialic acid content, or charge; it is generally more deficient in this acidic sugar than is hCG (Elliott et al., 1997, Endocrine 7:15-32). Normally-glycosylated hCG contains between 11 and 15 sialic acid residues, whereas ITA can contain between 8 and 19 sialic acid residues (Elliott et al., 1997, Endocrine 7:15-32). Sialic acid contributes to the biological activity of the molecule by protecting terminal galactose residues from liver galactose receptors, by increasing the circulating half-life of the molecule, and by stabilizing the hCG α-β dimer (Brand et al., 1980, Acta Endocrinologica 95:75-83; Rosa et al., 1984, J Clin Endocrinol Metab 59:1215-9; Van Hall et al., 1970, Endocrinology 89:11-15).
Increased proportions of ITA are produced in Down syndrome pregnancies (Cole et al., 1999, Clin Chem 45:2109-19). This is due to the failure of trophoblast cells to differentiate into syncytiotrophoblasts, leading to an accumulation of invasive cytotrophoblast cells in these pregnancies (Evain-Brion et al., 2000, Bull Acad Natl Med 184:1033-45; Frendo et al., 2000, J Clin Endocrinol Metab 85:3700-7; Massin et al., 2001, Placenta 22:S93-97).
Previous reports have demonstrated that ITA can be used as a marker for Down's syndrome pregnancies in the first and second trimester (Cole et al., 1998, Prenat Diagn 18:926-33; Cole et al., 1999, Clin Chem 45:2109-19; Cuckle et al., 1999, Prenat Diagn. 19:911-7; Strom et al., 2001, 51st Annual Meeting of the American Society of Human Genetics, San Diego, Calif., (Abstract 2839); Weinans et al., 2000, Prenat Diagn 20:976-8). One urine-based ITA study reports detecting 80% of Down syndrome cases with a 5% false-positive rate in the second trimester of pregnancy (Cole et al., 1999, Clin Chem 45:2109-19). In addition, when ITA was used in combination with the triple screen test (measures total hCG, unconjugated estriol, and α-fetoprotein), an even higher sensitivity marker was attained, detecting 96% of cases with a 5% false positive rate, or 94% of cases at a 3% false positive rate (Cole et al., 1999, Clin Chem 45:2109-19). A serum-based ITA study reported detecting 92% of Down syndrome cases with a 3% false positive rate (Lee et al., 2001, 51st Annual Meeting of the American Society of Human Genetics, San Diego, Calif., Abstract 160). First-trimester clinical trials indicated 60% detection of Down syndrome cases with a 5% false positive rate when using PAPP-A and hCG free-β subunit; however the addition of an ITA measurement raised the detection level to 81% with a 5% false positive rate (Strom et al., 2001, 51st Annual Meeting of the American Society of Human Genetics, San Diego, Calif., Abstract 2839).
Therefore, while screens that include detecting of ITA are vast improvements over previously used screening methods, increasing the positive rate and decreasing the false positive rate of the tests is desirable, particularly in the first trimester.